Regulated nucleic acid expression system

Abstract

The present invention provides nucleic acid constructs, expression systems, and methods relating to the regulation of gene expression. The invention may be applied to regulate the expression of any coding sequence of interest, including those coding for viral components necessary for the packaging of viral particles.

What is claimed is:  
     
       1. A packaging cell comprising: 
       a first nucleic acid construct comprising a nucleotide sequence encoding a first inducible promoter and a first product, wherein the first inducible promoter regulates expression of first product and the first inducible promoter is selected from the group consisting of a tetracycline promoter/operator, a retroviral long terminal repeat (LTR) and a steroid promoter region;  
       a second nucleic acid construct comprising a nucleotide sequence encoding a second inducible promoter and a second product, wherein the second inducible promoter is induced by the first product and the second product encodes a viral RNA binding protein; and  
       a third nucleic acid construct comprising a nucleotide sequence encoding a third inducible promoter and a viral gene product, wherein the third inducible promoter is induced by the second product.  
     
     
       2. The cell of  claim 1  wherein said first product is a transactivator of a tetracycline promoter/operator or a fusion protein comprising said transactivator. 
     
     
       3. The cell of  claim 1  wherein said second inducible promoter is a tetracycline promoter/operator. 
     
     
       4. The cell of  claim 1  wherein said viral RNA binding protein is a rev protein. 
     
     
       5. The cell of  claim 1  wherein said third inducible promoter is derived from a retroviral 5′ LTR. 
     
     
       6. The cell of  claim 1  wherein said first product is a tat protein or a chimeric protein comprising a tat protein. 
     
     
       7. The cell of  claim 1  which is stably transfected with said nucleic acid constructs. 
     
     
       8. The cell of  claim 1  further comprising a conditionally replicating viral vector and wherein said cell packages said vector. 
     
     
       9. The cell of  claim 8  wherein said vector is derived from HIV-1. 
     
     
       10. The cell of  claim 9  wherein said viral gene product is a VSV or Mokola virus G protein. 
     
     
       11. The cell of  claim 1  wherein the viral gene product is a viral envelope protein or a G protein. 
     
     
       12. The cell of  claim 11  further comprising an additional nucleic acid construct that encodes retroviral gag and pol proteins. 
     
     
       13. The cell of  claim 11  wherein said viral gene product is a G protein. 
     
     
       14. The cell of  claim 11  wherein said viral RNA binding protein is a rev protein. 
     
     
       15. The cell of  claim 11  further comprising a conditionally replicating viral vector and wherein said cell packages said vector. 
     
     
       16. The cell of  claim 15 , wherein said vector is derived from HIV-1. 
     
     
       17. A method of packaging a viral vector comprising culturing the cell of  claim 16  under conditions wherein said first nucleic acid construct expresses said first product. 
     
     
       18. The cell of  claim 1 , wherein said second product encodes rev and said third construct further comprises a nucleotide sequence encoding a rev responsive element (RRE). 
     
     
       19. A method of packaging a viral vector comprising culturing the cell of  claim 9  under conditions wherein said first nucleic acid construct expresses said first product.